embryonic stem cell

A stem cell derived from an embryos.
Specifically, embryonic stem cells are derived from embryos that develop
from eggs that have been fertilized in vitro
– in an in vitro fertilization clinic – and then donated for
research purposes with informed consent of the donors. They are not derived
from eggs fertilized in a woman's body. The embryos from which human embryonic
stem cells are derived are typically four or five days old and are a hollow
microscopic ball of cells called the blastocyst.
The blastocyst includes three structures: the trophoblast,
which is the layer of cells that surrounds the blastocyst; the blastocoel,
which is the hollow cavity inside the blastocyst; and the inner cell mass,
which is a group of approximately 30 cells at one end of the blastocoel.

Embryonic stem cells growth in the laboratory

Growing cells in the laboratory is known as cell culture.
Human embryonic stem cells are isolated by transferring the inner cell mass
into a plastic laboratory culture dish that contains a nutrient broth known
as culture medium. The cells divide and spread over the surface of the dish.
The inner surface of the culture dish is typically coated with mouse embryonic
skin cells that have been treated so they will not divide. This coating
layer of cells is called a feeder layer. The reason for having the mouse
cells in the bottom of the culture dish is to give the inner cell mass cells
a sticky surface to which they can attach. Also, the feeder cells release
nutrients into the culture medium. Recently, scientists have begun to devise
ways of growing embryonic stem cells without the mouse feeder cells. This
is a significant scientific advancement because of the risk that viruses
or other macromolecules in the mouse cells may be transmitted to the human
cells.

Over the course of several days, the cells of the inner cell mass proliferate
and begin to crowd the culture dish. When this occurs, they are removed
gently and plated into several fresh culture dishes. The process of replating
the cells is repeated many times and for many months, and is called subculturing.
Each cycle of subculturing the cells is referred to as a passage. After
six months or more, the original 30 cells of the inner cell mass yield millions
of embryonic stem cells. Embryonic stem cells that have proliferated in
cell culture for six or more months without differentiating, are pluripotent,
and appear genetically normal are referred to as an embryonic stem cell
line.

Once cell lines are established, or even before that stage, batches of them
can be frozen and shipped to other laboratories for further culture and
experimentation.

Laboratory tests used to identify embryonic
stem cells

At various points during the process of generating embryonic stem cell lines,
scientists test the cells to see whether they exhibit the fundamental properties
that make them embryonic stem cells. This process is called characterization.

As yet, scientists who study human embryonic stem cells have not agreed
on a standard battery of tests that measure the cells' fundamental properties.
Also, scientists acknowledge that many of the tests they do use may not
be good indicators of the cells' most important biological properties and
functions. Nevertheless, laboratories that grow human embryonic stem cell
lines use several kinds of tests. These tests include:

Growing and subculturing the stem cells for many months. This ensures
that the cells are capable of long-term self-renewal. Scientists inspect
the cultures through a microscope to see that the cells look healthy
and remain undifferentiated.

Using specific techniques to determine the presence of surface markers
that are found only on undifferentiated cells. Another important test
is for the presence of a protein called Oct-4, which undifferentiated
cells typically make. Oct-4 is a transcription factor, meaning that
it helps turn genes on and off at the right
time, which is an important part of the processes of cell differentiation
and embryonic development.

Examining the chromosomes under
a microscope. This is a method to assess whether the chromosomes are
damaged or if the number of chromosomes has changed. It does not detect
genetic mutations in the cells.

Determining whether the cells can be subcultured after freezing, thawing,
and replating.

Testing whether the human embryonic stem cells are pluripotent by 1) allowing
the cells to differentiate spontaneously in cell culture; 2) manipulating
the cells so they will differentiate to form specific cell types; or 3)
injecting the cells into an immunosuppressed mouse to test for the formation
of a benign tumor called a teratoma. Teratomas typically contain a mixture
of many differentiated or partly differentiated cell types – an
indication that the embryonic stem cells are capable of differentiating
into multiple cell types.

Stimulation of embryonic stem cells to differentiate

As long as the embryonic stem cells in culture are grown under certain conditions,
they can remain undifferentiated (unspecialized). But if cells are allowed
to clump together to form embryoid bodies, they begin to differentiate spontaneously.
They can form muscle cells, nerve cells, and many other cell types. Although
spontaneous differentiation is a good indication that a culture of embryonic
stem cells is healthy, it is not an efficient way to produce cultures of
specific cell types.

So, to generate cultures of specific types of differentiated cells –
heart muscle cells, blood cells, or nerve cells, for example – scientists
try to control the differentiation of embryonic stem cells. They change
the chemical composition of the culture medium, alter the surface of the
culture dish, or modify the cells by inserting specific genes. Through years
of experimentation scientists have established some basic protocols for
the directed differentiation of embryonic stem cells into some specific
cell types (see diagram.

If scientists can reliably direct the differentiation of embryonic stem
cells into specific cell types, they may be able to use the resulting, differentiated
cells to treat certain diseases at some point in the future. Diseases that
might be treated by transplanting cells generated from human embryonic stem
cells include Parkinson's disease,
diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's
muscular dystrophy, heart disease, and vision and hearing loss.